WO2022154288A1 - Monoclonal antibody specifically binding to protein kinase a catalytic subunit alpha, and use thereof for cancer diagnosis - Google Patents

Abstract

The present invention relates to a monoclonal antibody specifically binding to protein kinase A catalytic subunit alpha (PKACA), a use thereof, and the like. It has been identified that an antibody or an antigen-binding fragment thereof of the present invention specifically binds to a specific domain of PKACA and thus has remarkably greater affinity for a specific antigen of the present invention than a conventional PKA antigen, and actual cancer patients can be diagnosed. Therefore, by using an antibody or an antibody fragment of the present invention, PKACA and PKA autoantibodies can be specifically detected at the protein level so that cancer can be accurately diagnosed.

Description

Monoclonal antibody that specifically binds to the catalytic subunit alpha of protein kinase A and cancer diagnosis using the same

The present invention relates to a monoclonal antibody specifically binding to the catalytic subunit alpha (PKACA) of protein kinase A, and uses thereof.

This application claims priority based on Korean Patent Application No. 10-2021-0003891 filed on January 12, 2021, and all contents disclosed in the specification and drawings of the application are incorporated herein by reference.

Cancer is currently one of the diseases that cause the highest number of deaths worldwide, and the age of cancer onset is gradually decreasing while the average lifespan is gradually increasing, so the incidence of cancer is expected to further increase. Cancer occurs when cell division is not controlled normally due to genetic and environmental factors, and abnormal cell division occurs. However, if a reagent that can be easily diagnosed at an early stage of various cancers in the human body is developed, it is expected that the prognosis will be improved and ultimately the lifespan of mankind can be extended because early treatment can be carried out.

On the other hand, cyclic AMP (cAMP)-dependent protein kinase PKA (protein kinase A) is one of the most important enzymes for posttranscriptional modifications, cell proliferation, metabolism, gene induction, blood vessel formation, ion channel It plays important roles in various biological processes such as regulation and apoptosis. PKAs, including type I and type II, are predominantly intracellular enzymes, tetrameric enzymes composed of two regulatory units and two common catalytic subunits, and R dimers and two It is divided into free C sub-units.

In particular, it has been reported that various types of cancer cells release the C subunit of PKA out of the cell (Extracellular cAMP-dependent protein kinase A; ECPKA), thereby presenting PKA Ca in the culture medium of the cancer cells ( Proc Natl Acad Sci, 2000, 97(2): 835-40). In addition, in the above literature, it was confirmed that ECPKA activity was high in the serum of various cancer patients, and in contrast, it was confirmed that ECPKA activity did not appear relatively in the normal control group, suggesting the possibility of cancer diagnosis using ECPKA.

Under this background, the present inventors have made intensive efforts to develop a more improved cancer diagnosis method. As a result, the present invention was completed by developing a novel monoclonal antibody that specifically binds to a specific domain of human ECPKA in serum.

In order to solve the above problems and needs, the present inventors selected an antibody that specifically binds to the catalytic subunit alpha of protein kinase A, and confirmed through an experiment that the antibody can be used for actual cancer diagnosis. The present invention was completed.

Accordingly, an object of the present invention is an antibody or antibody that specifically binds to the catalytic subunit alpha of protein kinase A that recognizes a polypeptide selected from the group consisting of the amino acid sequence shown in SEQ ID NOs: 1 to 5 as an epitope. to provide a fragment of

Another object of the present invention is to provide a polynucleotide encoding the antibody or fragment of the antibody.

Another object of the present invention is to provide a recombinant expression vector comprising the polynucleotide.

Another object of the present invention is to provide a composition for diagnosing cancer comprising the antibody or antibody fragment.

Another object of the present invention is to provide a method for diagnosing ECPKA including the composition for diagnosing cancer.

Another object of the present invention is to provide a method for diagnosing ECPKA autoantibodies comprising the composition for diagnosing cancer.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Accordingly, the present invention provides an antibody or antibody fragment that specifically binds to the catalytic subunit alpha of protein kinase A that recognizes a polypeptide selected from the group consisting of the amino acid sequence shown in SEQ ID NOs: 1 to 5 as an epitope. provides

In one embodiment of the present invention, the antibody or fragment of the antibody is a heavy chain complementarity determining region (CDR) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7, amino acids selected from the group consisting of SEQ ID NOs: 8 and 9 a heavy chain variable region comprising a heavy chain CDR2 comprising the sequence, and a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11; and a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 17 It may include a light chain variable region comprising a light chain CDR3 comprising a, but is not limited thereto.

In another embodiment of the present invention, the antibody or antibody fragment comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 19; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 21, but is not limited thereto.

In another embodiment of the present invention, the antibody may be selected from the group consisting of IgG, IgA, IgM, IgE and IgD, but is not limited thereto.

In another embodiment of the present invention, the fragment of the antibody is selected from the group consisting of diabodies, Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv, dsFv, Fd, Fd' and scFv. may be, but is not limited thereto.

In addition, the present invention provides a polynucleotide encoding the antibody or fragment of the antibody.

In one embodiment of the present invention, the polynucleotide may include one or more sequences selected from the group consisting of nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 33, but is not limited thereto.

In another embodiment of the present invention, the polynucleotide may include one or more sequences selected from the group consisting of nucleotide sequences selected from the group consisting of SEQ ID NOs: 34 to 37, but is not limited thereto.

In addition, the present invention provides a recombinant expression vector comprising the polynucleotide.

In addition, the present invention provides a cell transformed with the recombinant expression vector.

In addition, the present invention comprises the steps of (a) transforming a host cell with the recombinant expression vector;

(b) culturing the transformed host cell to produce an antibody or antibody fragment; and

(c) provides a method for producing an antibody or antibody fragment that specifically binds to the catalytic subunit alpha of protein kinase A, comprising the step of obtaining an antibody or antibody fragment produced in a host cell.

In addition, the present invention provides a composition for diagnosing cancer comprising the antibody or antibody fragment.

In addition, the present invention provides the use of the antibody or fragment of the antibody for diagnosing cancer.

In addition, the present invention provides the use of the antibody or fragment of the antibody for producing a cancer diagnostic agent.

In one embodiment of the present invention, the cancer is colon cancer, colorectal cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, villous cancer, ovarian cancer , breast cancer, thyroid cancer, brain cancer, head and neck cancer, malignant melanoma, lymphoma, may be selected from the group consisting of aplastic anemia and blood cancer, but is not limited thereto.

In addition, the present invention provides a kit for diagnosing cancer comprising the composition for diagnosing cancer.

In addition, the present invention includes the step of detecting the catalytic subunit alpha of protein kinase A in a biological sample isolated from an individual suspected of cancer using the antibody or antibody fragment through an antigen-antibody reaction, diagnosis of cancer It provides a method of providing information for

In addition, the present invention comprises the step of detecting an autoantibody to the catalytic subunit alpha of protein kinase A through an antigen-antibody reaction in a biological sample isolated from an individual suspected of cancer using the antibody or antibody fragment. , to provide a method for providing information for cancer diagnosis.

In one embodiment of the present invention, the information providing method may further include, but is not limited to, determining that the patient is a cancer patient when the autoantibody level is higher than the level measured in the normal control group.

In another embodiment of the present invention, the subject suspected of cancer may be one or more selected from the group consisting of a patient who appears to be a normal person without any subjective symptoms, a patient suspected of having cancer, and a patient who is undergoing surgery or treatment for cancer, However, the present invention is not limited thereto.

In another embodiment of the present invention, the autoantibody may be produced by contacting the biological sample with a polypeptide selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 5, but is not limited thereto.

In addition, the present invention includes the step of detecting the catalytic subunit alpha of protein kinase A or an autoantibody thereto through an antigen-antibody reaction in a biological sample isolated from a subject suspected of cancer using the antibody or antibody fragment. To provide a method for diagnosing cancer.

In addition, the present invention includes the step of detecting the catalytic subunit alpha of protein kinase A or an autoantibody thereto through an antigen-antibody reaction in a biological sample isolated from a subject suspected of cancer using the antibody or antibody fragment. It provides a method of predicting the risk of developing cancer.

The antibody, or antigen-binding fragment thereof, of the present invention specifically binds to a specific domain of the catalytic subunit alpha of protein kinase A, thereby significantly increasing its affinity compared to the existing catalytic subunit alpha antigen of protein kinase A. It was confirmed that cancer patients can be diagnosed. Accordingly, the catalytic subunit alpha of protein kinase A can be specifically detected at the protein level in the future using the antibody or antibody fragment of the present invention to enable accurate diagnosis of cancer.

1 is a schematic diagram of a peptide conjugation mechanism for using a culture supernatant of a PKA clone cell as an indirect ELISA method for detecting a PKA antigen.

2 is a schematic diagram of an Indirect ELISA method for detecting PKACA antigen.

3a to 3e are ELISA results using the culture supernatant of PKACA and PKA clone cells. FIG. 3a is a PKA1 clone, FIG. 3b is a PKA2 clone, FIG. 3c is a PKA3 clone, FIG. 3d is a PKA4 clone, and FIG. ELISA results are shown.

Figure 4 is a schematic diagram of the sandwich ELISA method for the antigen-antibody detection method for PKACA-autoantibody.

Figure 5 shows the results of sandwich ELISA for measuring the binding force of PKA1-7 and PKA4-9 to the PKACA peptides 1 and 4 standard samples.

6 shows the results of sandwich ELISA using PKA1-7 and PKA4-9 for serum samples from prostate and breast cancer patients.

7 is a schematic diagram of a direct ELISA method for detecting a PKACA antigen-PKA autoantibody conjugate.

8 shows the results of detecting the PKACA antigen-PKA autoantibody conjugate from the serum samples of prostate and breast cancer patients through direct ELISA using PKA 1-7 and PKA 4-9.

Hereinafter, the present invention will be described in detail.

First, the present invention relates to an antibody or antibody fragment that specifically binds to the catalytic subunit alpha of protein kinase A that recognizes a polypeptide selected from the group consisting of the amino acid sequence shown in SEQ ID NOs: 1 to 5 as an epitope. provides

In the present invention, "catalytic subunit α of protein kinase A (PKACA)" is a major regulatory enzyme encoded by the PKACA gene in humans. This enzyme phosphorylates other proteins and substrates to change their activity. PKACA is found on chromosome 19 in humans. The most common form, called Cα1, is expressed throughout human tissues, and another transcript, Cα2, is mainly found in sperm cells and differs from Cα1 by only the first 15 amino acids. As described above, the antibody or fragments thereof according to the present invention can detect a partial domain region of PKACA while specifically detecting the C subunit (extracellular cAMP-dependent protein kinase A; ECPKA) of PKA released by cancer cells. have. The specific sequence of the PKACA is not particularly limited as long as it is known in the art as PKACA, and for example, the PKACA of the present invention is derived from human (homo sapiens) and is NCBI (Genbank) Accession No. It may include those known as NP_001291278.1, NP_002721.1, NP_997401.1, XP_016882437.1, and the like, but is not limited thereto.

As used herein, the term "epitope" refers to a protein determinant capable of specifically binding to an antibody. The epitope usually consists of a surface group of a molecule such as an amino acid or a sugar side chain, and generally has specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the conformational epitope is lost but not to the non-conformational epitope in the presence of a denaturing solvent. The epitope is an amino acid residue directly involved in binding (also referred to as an immunogenic component of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen binding peptide (i.e., amino acid residues). residues within the footprint of a specific antigen binding peptide).

Preferably, the binding site of the antibody of the present invention derived from the PKACA N-terminal sequence, as long as it is a contiguous region comprising the amino acid sequence shown in SEQ ID NOs: 1 to 5, the specific sequence is not particularly limited, usually SEQ ID NO: 11 to 52 including the amino acid sequence represented by 1 to 5, more preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acid sequences. The contiguous region comprising the amino acid sequence represented by SEQ ID NOs: 1 to 5 of the present invention may have substantially the same amino acid sequence as compared to the full length of the entire amino acid sequence or a fragment thereof. The amino acid sequence is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 compared to the full length of the protein sequence or a fragment thereof. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. A variant may be an amino acid sequence that is substantially identical to the full length of the amino acid sequence or a fragment thereof. The amino acid sequence is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 compared to the full length of the amino acid sequence or a fragment thereof. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

As used herein, the term "antibody" refers to a protein molecule serving as a receptor that specifically recognizes an antigen, including immunoglobulin molecules having immunological reactivity with a specific antigen, polyclonal antibody, monoclonal antibody, Includes both antigen-binding fragments (antibody fragments) of antibody molecules as well as complete antibody forms. The term also includes chimeric antibodies, humanized antibodies, and bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies and tetrabodies.

The "complete antibody" is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. The complete antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4.

As used herein, the term "antibody fragment" refers to a fragment having an antigen-antibody binding function in a complete antibody molecule, Fab, Fab', F(ab) ₂ , F(ab') ₂ , scFv, Fv , dsFv, diabody, Fd and Fd' and the like. The Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv (variable fragment) refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In a double chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in single chain Fv (scFv), the variable region of the heavy chain and the variable region of the light chain are generally covalently linked through a peptide linker. Alternatively, they may be directly linked at the C-terminus to form a dimer-like structure like a double-stranded Fv. A diabody (diabody) refers to a complex of two or more polypeptide chains or proteins, each comprising at least one VL and VH domain or a fragment thereof, and both domains are contained within a single polypeptide chain. In certain embodiments, the diabody comprises a molecule comprising an Fc or hinge-Fc domain. The polypeptide chains of these complexes may be the same or different, that is, the diabody may be a mono- or hetero-multimer.

Fragments of these antibodies can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the complete antibody with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), Preferably, it can be produced through genetic recombination technology.

The term "heavy chain" as used herein refers to a full-length heavy chain comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant domain domains CH1, CH2 and CH3, and fragments thereof means all In addition, as used herein, the term "light chain" refers to both a full-length light chain including a variable region domain VL and a constant region CL comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, and a fragment thereof.

As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such monoclonal antibody exhibits single binding specificity and affinity for a specific epitope. Typically, immunoglobulins have heavy and light chains, each heavy and light chain comprising a constant region and a variable region (these regions are also known as domains). The variable regions of the light and heavy chains comprise three variable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions (FRs). The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are called sequentially CDR1, CDR2, CDR3, typically starting from the N-terminus, and are also identified by the chain in which the specific CDR is located.

As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of an immunoglobulin heavy chain and light chain (Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). The heavy (CDRH1, CDRH2 and CDRH3) and light chains (CDRL1, CDRL2 and CDRL3) each contain three CDRs, which provide the key contact residues for the binding of the antibody to the antigen or epitope.

As used herein, the term "chimeric antibody" is an antibody in which the variable region of a heterologous antibody and the constant region of a human antibody are recombined by DNA recombination technology for humans, such as mice and chickens, Since the immune response of the chimeric antibody is greatly improved compared to an antibody that is heterogeneous to humans such as mice and chickens, it can be used clinically.

As used herein, the term "humanized antibody" refers to an antibody in which all or part of the CDR sequences of a monoclonal antibody heterologous to humans, such as mice and chickens, are grafted into a human antibody. For example, a humanized variable region may be prepared by recombination of CDRs of a chicken or mouse monoclonal antibody with FR derived from a human antibody, and it may be prepared by recombination with the constant region of a desired human antibody, but is not limited thereto. In addition, when only the chicken or mouse-derived CDRs are transplanted, the affinity of the humanized antibody is lowered, so FR amino acid residues, which can be considered to affect the three-dimensional structure of the CDRs, are substituted with amino acids of the chicken or mouse antibody. may promote, but is not limited thereto.

The antibody or antigen-binding fragment thereof of the present invention includes all mutants that achieve the desired effect of the present invention through mutation, such as one or more substitutions, deletions, inversions or translocations, in the antibody defined by the above sequence. .

In the present invention, the antibody or antibody fragment comprises a heavy chain complementarity determining region (CDR) 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9 a heavy chain variable region comprising a heavy chain CDR2 and a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11; and a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 17 It may include a light chain variable region comprising a light chain CDR3 comprising a, but is not limited thereto.

In the present invention, the antibody or antibody fragment comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 19; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 21, but is not limited thereto.

In the present invention, the antibody may be selected from the group consisting of IgG, IgA, IgM, IgE and IgD, but is not limited thereto.

In the present invention, the antibody fragment may be selected from the group consisting of diabody, Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv, dsFv, Fd, Fd' and scFv, but this It is not limited.

In addition, the present invention provides a polynucleotide encoding the antibody or fragment of the antibody.

Polynucleotides herein may also be described as oligonucleotides or nucleic acids, which are produced using DNA molecules (eg, cDNA or genomic DNA), RNA molecules (eg, mRNA), nucleotide analogues. DNA or RNA analogues (eg, peptide nucleic acids and non-naturally occurring nucleotide analogues) and their hybrids.The polynucleotide is single-stranded or double-stranded. It can be double stranded.

In the present invention, the polynucleotide may include one or more sequences selected from the group consisting of nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 33, but is not limited thereto. The polynucleotide may have a nucleotide sequence that is “substantially identical to” the nucleotide sequence. The base sequence is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 compared to the full length of the gene sequence or a fragment thereof. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

"Substantially identical," as used herein, means that the first and second sequences are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, over a region of 95, 100, 180, 270, 300, 360, 450, 540 or more nucleotides or amino acids or for nucleic acids, at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In the present invention, the polynucleotide may include one or more sequences selected from the group consisting of nucleotide sequences selected from the group consisting of SEQ ID NOs: 34 to 37, but is not limited thereto. The polynucleotide may have a nucleotide sequence that is “substantially identical to” the nucleotide sequence.

The polynucleotide encoding the antibody or fragment thereof of the present invention can be obtained by methods well known in the art. For example, based on the DNA sequence or the corresponding amino acid sequence encoding a part or all of the heavy and light chains of the antibody, an oligonucleotide synthesis technique well known in the art, for example, the polymerase chain reaction (PCR) method, etc. It can be synthesized using

In addition, the present invention provides a recombinant expression vector containing the polynucleotide, and cells transformed with the vector.

In the present invention, "recombinant" can be used interchangeably with "genetic manipulation", and molecular cloning experimental techniques such as modifying, cutting, and linking genes are used in the natural state. It refers to the production of a non-existent form of a gene. In the present invention, "expression" means that a protein or nucleic acid is produced in a cell.

In the present invention, "recombinant expression vector" refers to a vector capable of expressing a desired protein or nucleic acid (RNA) in a suitable host cell, and essential control operably linked so that the polynucleotide (gene) insert can be expressed. Refers to a genetic construct comprising an element. "Operably linked" means a nucleic acid expression control sequence in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function, and the gene is It means connected so that it can be expressed. The "expression control sequence" refers to a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a specific host cell. Such regulatory sequences include a promoter for effecting transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, a sequence regulating the termination of transcription and translation, an initiation codon, a stop codon, polyadenylation signals and enhancers.

The type of the recombinant expression vector of the present invention is not particularly limited as long as it is a vector commonly used in the fields of cloning and antibody production, and examples thereof include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. does not The plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24 and YCp50), and the like, As the virus, animal viruses such as retroviruses, adenoviruses or vaccinia viruses, insect viruses such as baculoviruses, etc. may be used. A vector of the pComb3 family commonly used for phage display may be used, and a vector commonly used to express a protein in mammalian cells, for example, a vector such as pcDNA or pVITRO, may be used to express the antibody in mammalian cells. have.

Accordingly, the recombinant expression vector according to the present invention is a gene construct operably linked so that a polynucleotide encoding an antibody or a fragment thereof comprising heavy and light chains having the above CDRs or VH and VL configurations can be expressed in an appropriate host cell. means

The cell type of the present invention is not particularly limited as long as it can be used to express a polynucleotide encoding an antibody or fragment thereof contained in the recombinant expression vector of the present invention. Cells (host cells) transformed with the recombinant expression vector according to the present invention are prokaryotes (eg, E. coli), eukaryotes (eg yeast or other fungi), plant cells (eg, tobacco or tomato plants). cells), animal cells (eg, human cells, monkey cells, hamster cells, rat cells, mouse cells, insect cells, or hybridomas derived therefrom. For example, the cells may be cells derived from mammals including humans, for example, HEK293T, 293F cells, etc. may be used.

The term 'transformation' refers to a change in the genotype of a host cell by introduction of an exogenous polynucleotide, and means that the exogenous polynucleotide is introduced into a host cell regardless of the method used for the transformation. An exogenous polynucleotide introduced into a host cell may be maintained integrated into the genome of the host cell or maintained without integration, and the present invention includes both.

Recombinant expression vector capable of expressing the polypeptide of the antibody or antibody fragment according to the present invention is a method known in the art, for example, but not limited to, transient transfection (transient transfection), microinjection, transduction ( transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection transfection), electroporation, a gene gun, and a known method for introducing a nucleic acid into a cell can be introduced into a cell for producing an antibody or a fragment thereof and transformed. The cells transformed with the recombinant expression vector according to the present invention produce heavy chains, light chains, or antibody fragments of the antibody according to the present invention.

In addition, the present invention comprises the steps of (a) transforming a host cell with the recombinant expression vector;

(b) culturing the transformed host cell to produce an antibody or antibody fragment; and

(c) provides a method for producing an antibody or antibody fragment that specifically binds to the catalytic subunit alpha of protein kinase A, comprising the step of obtaining the antibody or antibody fragment produced in the host cell.

Step (a) is a step of transforming a host cell for producing an antibody or fragment thereof according to the present invention with a recombinant expression vector to which a polynucleotide encoding the antibody or fragment is operably linked.

A person skilled in the art can perform this step by selecting an appropriate transformation method as described above according to the selected host cell and the type of recombinant expression vector. The recombinant expression vector containing the nucleotide sequences of the heavy chain and the light chain may be co-transformed in the same host cell so that the heavy and light chains can be expressed in a single cell, or the recombinant expression vector containing the nucleotide sequences of the heavy and light chains can be separated from each other. It is also possible to transform the host cell of a so that the heavy and light chains are expressed separately.

Step (b) is a step of culturing the transformed host cell to produce a polypeptide of the heavy chain, light chain or antibody fragment of the antibody according to the present invention from the recombinant expression vector introduced into the host cell.

A person of ordinary skill in the art can appropriately select the composition of the medium for culturing the selected host cell, culture conditions, culture time, and the like. The antibody molecule produced in the host cell may be accumulated in the cytoplasm of the cell, secreted to the outside of the cell or culture medium by an appropriate signal sequence, or targeted by periplasm or the like. In addition, it is preferable that the antibody according to the present invention has a protein refolding (refolding) and functional structure (conformation) using a method known in the art to maintain the binding specificity to PKACA. In addition, when producing an antibody in the form of IgG, the heavy and light chains are expressed in separate cells, and the heavy and light chains are contacted in a separate step to form a complete antibody, or the heavy and light chains are expressed in the same cell. It can also allow the formation of complete antibodies inside the cell.

Step (c) is a step of obtaining an antibody or fragment thereof produced in a host cell. A person skilled in the art can appropriately select and control the obtaining method in consideration of the characteristics of the antibody or fragment thereof produced in the host cell, the characteristics of the host cell, the expression method, or whether the polypeptide is targeted. For example, the antibody or fragment thereof secreted into the culture medium can be recovered by a method such as obtaining a culture medium for host cells and centrifuging to remove impurities. If necessary, the cells may be lysed in a range that does not affect the functional structure of the antibody or fragment thereof in order to release and recover the antibody present in a specific organelle or cytoplasm outside the cell. In addition, the obtained antibody may be further subjected to a process of further removing impurities and concentrating through a method such as chromatography, filtration using a filter, dialysis, or the like.

In addition, the present invention provides a composition for diagnosing cancer comprising the antibody or antibody fragment.

In the present invention, the cancer is colon cancer, colorectal cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, villous cancer, ovarian cancer, breast cancer, thyroid cancer , may be selected from the group consisting of brain cancer, head and neck cancer, malignant melanoma, lymphoma, aplastic anemia, and blood cancer, preferably prostate cancer or breast cancer, but is not limited thereto.

In the present invention, the term “diagnosis” refers to confirming the presence, severity (symptoms), and/or characteristics of a pathological condition. As used herein, "diagnosis of cancer" refers to confirming the pathological state of cancer, such as whether or not cancer occurs, the extent of the cancer site, the location of cancer (cancer cells), and the degree of colorectal cancer, and a more accurate diagnosis For this purpose, it is important to accurately and quickly distinguish cancer cells or cancer cells and/or tissues from normal cells or normal tissues.

In addition, the composition for diagnosis of cancer according to the present invention contains, in addition to the antibody or antibody fragment according to the present invention, elements commonly used to detect a protein as an antibody, such as a secondary antibody, a substrate for the color reaction of the secondary antibody, and a cofactor. may additionally include.

In addition, the present invention provides a kit for diagnosing cancer comprising the composition for diagnosing cancer.

The composition and cancer are the same as described above. In addition, the cancer diagnosis kit may further include a composition, solution, or device having one or more other components suitable for the analysis method.

In the present invention, the cancer diagnostic kit may be used as a means for detecting an anti-human PKACA autoantibody, but is not limited thereto. That is, the kit may include a PKA or PKACA antigen known in the art as an antigen of an autoantibody, and may include the PKACA-derived peptide of the present invention. The PKACA is a known PKACA or 80%, 90%, 95%, 96%, 97%, 98% or 99%, etc. It may be a variant having homology to, and deletion, substitution, or addition of some sequences from a known sequence may also be included within the scope of the present invention. For example, the PKACA-derived peptide may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5, but is not limited thereto.

In addition, the present invention includes the step of detecting the catalytic subunit alpha of protein kinase A in a biological sample isolated from an individual suspected of cancer using the antibody or antibody fragment through an antigen-antibody reaction, diagnosis of cancer It provides a method of providing information for

In addition, the present invention comprises the step of detecting an autoantibody to the catalytic subunit alpha of protein kinase A through an antigen-antibody reaction in a biological sample isolated from an individual suspected of cancer using the antibody or antibody fragment. , to provide a method for providing information for cancer diagnosis.

In the present invention, the method for providing information may further include determining that the patient is a cancer patient when the level of the protein kinase A catalytic subunit alpha or autoantibody thereto is higher than that measured in a normal control group. It is not limited.

In the present invention, the subject suspected of cancer may be one or more selected from the group consisting of a patient who appears to be a normal person without any subjective symptoms, a patient suspected of having cancer, and a patient undergoing or after surgery for cancer, but is limited thereto not.

In the present invention, the autoantibody may be produced by contacting the biological sample with a polypeptide selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 5, but is not limited thereto.

In the present invention, the autoantibody may be directed against PKA or PKACA antigen known in the art, but is not limited thereto.

As used herein, the term "autoantibody" refers to an antibody that targets an antigen derived from the same individual. Based on the fact that serum, whole blood, plasma or urine levels of anti-human PKACA autoantibodies in various cancer patients are high, it is a technology that can detect early cancer with high sensitivity and specificity that can be applied to actual clinical practice. has not been developed to date.

As used herein, the term "biological sample" includes tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells and bacterial expression systems. However, the present invention is not limited thereto. The presence or absence of the PKACA protein or cancer can be confirmed by reacting these biological samples with the antibody of the present invention in an manipulated or unmanipulated state.

As used herein, the term "antigen-antibody complex" refers to a combination of a PKACA protein antigen in a sample and a monoclonal antibody according to the present invention that recognizes it, and the formation of such an antigen-antibody complex is performed by a colormetric method, electrochemical method Any method selected from the group consisting of electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and scintillation counting method can be detected with However, it is not necessarily limited to these, and various applications and applications are possible.

In the present invention, various markers can be used to detect the antigen-antibody complex. Specific examples may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioactive isotopes, but are not necessarily limited thereto.

Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, β-latamase, and the like, and the fluorescent substance is fluorescein. Phosphorus, quantum dots, Eu ³⁺ , Eu ³⁺ chelate or cryptate, etc., and the ligand includes a biotin derivative, and the luminescent material includes an acridinium ester, an isoluminol derivative, and the like. , and microparticles include colloidal gold, colored latex, and the like, and radioactive isotopes include ⁵⁷ Co, ³ H, ¹²⁵ I, ¹²⁵ I-Bonton Hunter reagents, and the like.

Preferably, the antigen-antibody complex can be detected using enzyme immunosorbent assay (ELISA). The enzyme immunosorbent assay (ELISA) includes a direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, and an indirect ELISA using a labeled secondary antibody recognizing a capture antibody in a complex of an antibody recognizing an antigen attached to a solid support. , use another labeled antibody that recognizes the antigen in the complex of the antibody and antigen attached to the solid support, or the antibody attached to the solid recognizes the antigen in the antigen-antibody complex and uses a labeled secondary antibody that recognizes the antibody Various ELISA methods, such as direct sandwich ELISA using a solid support, and indirect sandwich ELISA using a labeled secondary antibody recognizing the antibody after reacting with another antibody recognizing the antigen in a complex of an antibody attached to a solid support and the antigen, are included.

The monoclonal antibody may have a detection label, and when it does not have a detection label, these monoclonal antibodies can be captured and confirmed by treating another antibody having a detection label.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Synthesis of Protein Kinase A (PKA, Protein Kinase A) Catalyst Subunit Alpha Detection Peptide

Antigen for detecting protein kinase A catalytic subunit alpha (PKACA) analyzes the amino acid sequence information and three-dimensional structure of PKACA to determine a region with high antigenic index and high hydrophilicity, and selects a different position to enable sandwich binding in position. It was synthesized and used. Peptides were prepared using the Fmoc solid-phase peptide synthesis method, confirmed through MS and Reverse-HPLC, and purified to synthesize PKACA peptides (see Table 1).

Specifically, in the case of the Fmoc solid-phase peptide synthesis method, the coupling-wash-deprotection-wash cycle is repeated to remove excess reagent. Through this method, peptides are synthesized from the C-terminus to the N-terminus.

First, Rink Amide MBHA resin (called Fmoc Linker MBHA resin) was expanded by adding DCM and leaving it at room temperature for 30 minutes. Then DCM was removed and washed 3 times with DMF. The amino group fmoc was deprotected with piperidine, washed with DMF, HBTU was added, and then reacted with the carbonyl group of the amino-protected amino acid Fmoc-Trp(Boc)-OH for 30 minutes. The above process was repeated until the desired peptide chain was formed. A Kaiser test was used to detect free ammonia, and if the color did not change at this time, the next step was performed.

After the previous reaction was completed, the synthesized peptide was separated from the resin using reagent K TFA/thioanisole/water/phenol/EDT (82.5%/5%/5%/5%/2.5%). Thereafter, cold DEE was added to precipitate the product, washed with cold DEE, and vacuum dried to obtain a peptide.

M.W. of the synthesized peptide was measured through GC-MS, and the purity was confirmed at 220 nm wavelength by Reverse-HPLC using a C18 column.

Peptide name	Sequence information (N terminus → C terminus)	SEQ ID NO:
PKA1	EQESVKEFLAKAC	One
PKA2
KEDFLKKWESPAQNTAC	2
PKA3
SGKVRFPSHFSSDLC	3
PKA4
SNFDDYEEEEIRVC	4
PKA5
EEIRVSINEKC	5

Example 2. Synthesis of hybridoma producing PKACA detection antibody

As shown in FIG. 1, the synthesized peptide was immunized with BSA, a KLH (carrier protein), and the peptides synthesized in Example 1, respectively, using a Sulfo-SMCC crosslinker, and B lymphocytes and myeloma collected from the mouse spleen. The hybridoma cells fused with the cells were cultured to synthesize an anti-PKACA antibody specific for a peptide, and an antibody having a high titer to PKA was selected for screening by ELISA.

Specifically, 100 μg of antigen per BALB/c mouse was mixed with an adjuvant and injected using Abclone's MANI method (Monoclonal Antibody Next Innovation). 3 days before fusion, 100 μg of antigen was mixed with 1X PBS to increase the immune response, and 3 days later, B lymphocytes were isolated from the mouse spleen and mixed with SP2/0-Ag14 (ATCC, CRL-1581), a myeloma cell, and PEG- 1500 (Roche, 783641) solution was used for fusion. The fused cells were cultured in a medium containing hypoxanthin, aminopterine, and thymidine (HAT supplement, Sigma-Aldrich, H0262), and cells in which myeloma cells and B lymphocytes were fused ( hybridoma) was selectively cultured (only transformed hybridoma cells survived HAT medium selection). The obtained hybridoma cells were selected by using an ELISA method to identify and select a cell type that produces an antibody with a high degree of reactivity with the antigen (OD>0.5).

As shown in the indirect ELISA method in FIG. 2 , 100 ng of PKA antigen was diluted in 100 μl of coating buffer (0.1 M sodium carbonate buffer) in a 96-well ELISA plate (Corning, 3590), and incubated at 37° C. for 1 hour after loading. After incubation, 300 μl/well of 1X PBST solution was added and washed three times, and then blocked with 200 μl of 2% skim milk for 30 minutes at room temperature, 300 μl/well of 1X PBST solution was added and washed 3 times. Then, 100 μl/well of the hybridoma cell culture supernatant (sup.; solution containing the capture antibody) was loaded and incubated at 37° C. for 1 hour. Then, after washing in the same manner as in the previous procedure, anti-mouse IgG-HRP (Abclon, Abc5001) was diluted 1:20,000 as a secondary antibody, loaded at 100 μl/well, and incubated at 37° C. for 1 hour. Then, after washing in the same manner as in the previous process, 100 μl/well of TMB solution (Biofx, TMBC-1000) was loaded at a time, followed by color development for 5 minutes, and 100 μl/well of 1N sulfuric acid was loaded for reaction. was terminated. The absorbance was then measured at 450 nm. At this time, through ELISA, positive cells that specifically react with antigens are hybridized to produce antigen-responsive antibodies by repeating the process of separating positive and negative cells using the limiting dilution method (cloning). Hybridomas were produced. The produced hybridoma cells were frozen by 1X10 ⁶ cell/ml and stored in a liquid nitrogen tank.

Example 3. Selection of antibodies in PKACA clone cell supernatant

Invitrogen's PKACA (hereinafter referred to as PKA) recombinant human protein was used as an antigen, and the PKA clone cell culture supernatant prepared in Example 2 was used as an antibody. was selected.

Specifically, in a 96-well immunoplate, PKA recombinant human protein as an antigen was diluted with 0.1% BSA+0.05% Tween20 to make 1 ng/μl, then coated at 100 μl/well, and incubated at 4° C. for 19 hours. Then, 300 μl/well of 1X PBST was added, washed 3 times, and 100 μl/well of the PKA clone cell culture supernatant prepared in Example 2 was used as an antibody, and incubated at 37° C. for 2 hours. Then, it was washed with 1X PBST in the same manner as in the previous procedure, and then, 0.2ng/μl of anti-mouse IgG-HRP was loaded at 100μl/well, and incubated at 37°C for 2 hours. Then, 100 μl/well of TMB was loaded, wrapped in foil, darkened, and color developed at room temperature for 10 minutes. 0.5MH ₂ SO ₄ After stopping the reaction by loading 100 μl/well, the absorbance value was measured at 450 nm.

As shown in Figures 3a to 3e, antibodies PKA1-7 (4F9), 1-6 (7E12), 4-9 (15G11), 5-10 that are the top four PKA 1 to 5 Clone with high intensity at O.D@450nm (5H2) was selected.

Example 4. Optimization of PKA antigen coating amount using Indirect ELISA method

Indirect ELISA test was performed using the PKA recombinant human protein purchased from Invitrogen as the antigen and the upper supernatant of the PKA clone cell culture supernatant identified in Example 3 as the antibody, and the optimal coating amount was determined through the O.D@450nm value. was analyzed.

Specifically, 100 μl of 0.5, 1, and 2 ng/μl of PKA recombinant human protein was loaded onto a 96-well immunoplate, and then incubated at 4° C. for 19 hours. Then, 1X PBST was added at 300 μl/well, washed 3 times, and then blocked using 0.1% BSA+0.05% Tween20 and incubated at 37° C. for 2 hours. Then, 300 μl/well of 1X PBST was added and washed 3 times, and then 100 μl/well of the supernatant of the PKA clone cell culture supernatant identified in Example 3 was used as the primary antibody and loaded at 100 μl/well, for 2 hours. Incubated at 37°C. Then, it was washed with 1X PBST in the same manner as in the previous procedure, and then, 0.2 ng/μl of anti-mouse IgG-HRP was loaded at 100 μl/well, and incubated at 37° C. for 2 hours. Then, 100 μl/well of TMB was loaded, wrapped in foil, darkened, and color developed at room temperature for 10 minutes, followed by 0.5MH ₂ SO ₄ After stopping the reaction by loading 100 μl/well, the OD value was measured at 450 nm. The highest intensity was confirmed at OD@450nm, which was performed by loading PKA recombinant human protein by 200ng/well, and then the experiment was performed under the corresponding conditions.

Example 5. Confirmation of interaction between each PKACA peptide and the top 4 PKA clone cell culture supernatant (antibody)

Binding of antibodies to each PKACA peptide (antigen) in anti-PKA1-7(4F9), 1-6(7E12), 4-9(15G11), 5-10(5H2) supernatant of the top 4 PKA clone cell cultures To confirm the degree, values at O.D@450nm were measured using a sandwich ELISA method.

Specifically, 100 μl of the top 4 PKA clone cell culture supernatant was loaded into a 96-well immunoplate and cultured at 4° C. for 19 hours. Then, 300 μl/well of 1X PBST was added, washed three times, and blocked using 0.1% BSA+0.05% Tween20 and incubated at 37° C. for 2 hours. Then, 300 μl/well of 1X PBST was added and washed three times, and then the peptide prepared in Example 1 was diluted with 0.1% BSA+0.05% Tween20 to make 2ng/μl. Each of the supernatant loading wells were loaded at 100 μl/well, and incubated at 37° C. for 2 hours. Then, it was washed with 1X PBST in the same manner as in the previous procedure, and 100 μl of the PKA α/β CAT antibody was diluted 10,000:1 using 0.1% BSA+0.05% Tween20, loaded at a time, and incubated at 37°C for 2 hours. did. Then, 300 μl/well of 1X PBST was added and washed 3 times, and then 100 μl of rabbit anti-mouse IgG HRP (@0.8 mg/ml) was diluted at 10,000:1 using 0.1% BSA+0.05% Tween20 and loaded at 100 μl, Incubated at 37° C. for 2 hours. Then, 300 μl/well of 1X PBST was added and washed 3 times, and then 100 μl/well of TMB was loaded, wrapped in foil, and darkened to develop color at room temperature for 10 minutes. And 0.5MH ₂ SO ₄ After stopping the reaction by loading 100 μl/well, the absorbance value was measured at 450 nm.

As shown in Table 2, it was confirmed that the intensity was different at O.D@450nm depending on the clone. Among the PKACA antigen peptides, we tried to select a sample with a large absorbance value for PKA 1, 3, and 4 and a small error range. Among the many clones, PKA1-7 and 4-9 were considered to show the most suitable antigen-binding ability, so it was confirmed and purified. and detection experiments were carried out.

No.	PKA Clone cell culture sup.	Antigen	O.D@450nm	Error
One	1-6 (7E12)	PKA peptide1	3.117	1.390
2	1-7 (4F9)	PKA peptide1	3.250	0.280
3	2-1 (3G2)	PKA peptide2	3.136	3.6582
4	2-2 (2D5)	PKA peptide2	3.329	3.463
5	3-1 (1A6)	PKA peptide3	2.971	3.561
6	3-5 (4E7)	PKA peptide3	2.998	1.010
7	4-4 (11E12)	PKA peptide4	3.005	2.359
8	4-9 (15G11)	PKA peptide4	3.381	0.038
9	5-1 (3A9)	PKA peptide5	3.527	2.377
10	5-10 (5H2)	PKA peptide5	3.634	1.944

Example 6. Confirmation of utility of existing PKA antigen using sandwich ELISA method

PKA recombinant human protein (Invitrogen_PKA Ag) purchased from Invitrogen and the PKA clone cell culture supernatant were used as antigens using the sandwich ELISA method shown in FIG. 4 to confirm their usefulness, and compared with the PKA peptide prepared in Example 1.

Specifically, 100 μl/well of the top 10 supernatants having high OD@450 nm intensity confirmed in Example 5 were loaded onto a 96-well immunoplate, and incubated at 4° C. for 19 hours. 1X PBST was loaded at 300 μl/well, washed three times, and blocked by loading 0.1% BSA+0.05% Tween20 at 300 μl/well, and incubated at 37° C. for 2 hours. Then, 1X PBST was loaded at 300 μl/well, washed 3 times, diluted with 0.1% BSA+0.05% Tween20 to make 2ng/μl of Invitrogen_PKA Ag, and then loaded at 100 μl/well and incubated at 37° C. for 2 hours. . Then, 1X PBST was loaded at 300 μl/well, washed 3 times, and 0.1% BSA+0.05% Tween20 was used to dilute rabbit anti-mouse IgG H&L (HRP) (ab6728, 2mg/ml) at a 1:10,000 ratio of 0.2 ng/μl was loaded at 100 μl/well, and incubated at 37° C. for 2 hours. Then, 300μl/well of 1X PBST was loaded and washed 3 times, and 100μl/well of TMB was added for 10 minutes for color development, followed by 0.5MH ₂ SO ₄ After stopping the reaction by loading 100 μl/well, the OD value was measured at 450 nm.

Most of the supernatant had an absorbance of 0.5 or more, confirming its usefulness as an antigen of Invitrogen_PKA for the culture supernatant of the PKA clone cell synthesized in Example 2.

In addition, as a result of comparing the absorbance of the PKACA peptide synthesized in Example 1 with the antigen, it was confirmed that the strength of the PKACA peptide synthesized in Example 1 was higher in the reaction with the PKA clone cell culture supernatant.

Example 7. Identification of the PKA antibody sequence of the present invention

For the variable region sequence analysis of the selected antibody library, RNA extraction (prep.) was performed from each hybridoma cell line, and after cDNA synthesis, the light chain and heavy chain variable region genes were amplified by PCR. The results are shown in Table 3 below.

	VL			VH
Clone	CDR1	CDR2	CDR3	CDR1	CDR2	CDR3
PKA 1-7	SASSSVSYMY	DTPNLAS	QQWSSYPFT	TYAMN	RIRSKSNNYATYYADSVKD	PYYYYGSSYMDY
PKA 4-9	TASSSVSSSYLH	STSNLAS	HQFHRSPFT	HYAM	VISIYYDNTNYNQKFKG	SGSTMITGFAY

	VL	VH
Clone
PKA 1-7	DIVMTQSPAIMSASPGEKVTMTC SASSSVSYMY WYQQKPGSSPRLLIY DTPNLAS GVPVRFSGSGSGTSYSLTISRMEAEDAATYYC QQWSSYPFT FGSGTKLELK	EVQRVESGGGLVQPKGSLKLSCAVSGFTFK TYAMN WVRQAPGKGLEWVA RIRSKSNNYATYYADSVKD RFTISRDDSQSMLYLQMNNLKTEDTAMYYCVS PYYYYGSSYMDY WGQGTTVTVSS
PKA 4-9	DVVMTQSPAIMSASLGERVTMTC TASSSVSSSYLH WYLQKPGSSPKLWIY STSNLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC HQFHRSPFT FGSGTKLEIK	EVQLQQSGPELVRPGESVRISCKGSGYTFT HYAMH WVKQSHAKSLEWIG VISIYYDNTNYNQKFKG KATMTVDKSSSTAYMELARLTSEDSAIYYCAR SGSTMITGFAY WGQGTLVTVSA

* CDR: bold

Example 8. Confirmation of binding affinity of the antibody of the present invention

Using the PKA 1-7 and 4-9 hybridoma culture supernatant (the secondary antibody is anti-mIgG-HRP), the binding ability to each antigen was confirmed by ELISA. ELISA conditions are shown in Table 5 below.

As shown in FIG. 5 , it was confirmed that the IgG antibody in the supernatant of the present invention had excellent binding ability to each antigen.

Example 9. Confirmation of cancer diagnostic effect of the antibody of the present invention (1)

In order to confirm the cancer diagnostic effect of the PKA antibody of the present invention, a sandwich ELISA was performed using the PKA antibody of the present invention on serum samples from prostate cancer, breast cancer and healthy people.

Specifically, 500 μL of antibodies PKA 1-7 and PKA 4-9 (0.1, 1.0, 2.0, 2.0, 10.0 μg/mL) were dissolved in 10 mL of carbonate and bicarbonate buffer (pH 9.5) and 96-well plate for surface coating. (100 μL/well). Incubated overnight at 4°C. Then, 300 μL/well blocking solution [1% BSA in PBS (300 μL/well)] was added and incubated at 37° C. for 2 hours. The blocking wells were washed with PBST (3X) and serum samples (10 μL) were added to 100 μL/well to prepare a 10 mL solution of 0.1% BSA + 0.05% Tween20 (1 μL/10 mL). After incubation with the antigen at 37°C for 2 hours, it was washed 3 times with PBST, and a secondary antibody, goat anti-human IgG HRP (1μg/mL), was added to the same solution at a concentration of 1μL/10mL and incubated at 37°C for 2 hours. The culture plate was washed 3 times with PBST and TMB substrate solution was added at 100 μL/well and placed at 37° C. for 15 minutes. The reaction was stopped by adding 0.5MH ₂ SO ₄ (100 μL/well) and absorbance was measured at 450 nm wavelength.

As shown in FIG. 6 , it was confirmed that the antibodies PKA 1-7 and PKA 4-9 of the present invention significantly differentiate between normal controls and cancer patients, so it is expected that the antibodies of the present invention can be used for cancer diagnosis. .

Example 10. Confirmation of cancer diagnostic effect of the antibody of the present invention (2)

In order to confirm the cancer diagnostic effect of the PKA antibody of the present invention, detection analysis was performed on serum samples from prostate cancer, breast cancer, and healthy people. As shown in FIG. 7 , the PKA antigen-PKA autoantibody conjugate was subjected to direct ELISA using the PKA antibody of the present invention using the direct ELISA method.

Specifically, 500 μL of antibodies PKA 1-7 and PKA 4-9 (0.1, 1.0, 2.0, 2.0, 10.0 μg/mL) were dissolved in 10 mL of carbonate and bicarbonate buffer (pH 9.5) and 96-well plate for surface coating. (100 μL/well) incubated overnight at 4°C. Then, 300 μL/well blocking solution [1% BSA in PBS (300 μL/well)] was added and incubated at 37° C. for 2 hours. The blocking wells were washed with PBST (3X) and serum samples (10 μL) were added to 100 μL/well to prepare a 10 mL solution of 0.1% BSA + 0.05% Tween20 (1 μL/10 mL). Serum samples were incubated at 37°C for 2 hours, washed 3 times with PBST, and a secondary antibody, goat anti-human IgG HRP (1μg/mL), was added to the same solution at a concentration of 1μL/10mL and incubated at 37°C for 2 hours. . The culture plate was washed 3 times with PBST and TMB substrate solution was added at 100 μL/well and placed at 37° C. for 15 minutes. The reaction was stopped by adding 0.5MH ₂ SO ₄ (100 μL/well) and absorbance was measured at 450 nm wavelength.

As shown in FIG. 8 , it was confirmed that the antibodies PKA 1-7 and PKA 4-9 of the present invention significantly differentiate between normal controls and cancer patients, so it is expected that the antibodies of the present invention can be used for cancer diagnosis. .

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

The antibody, or antigen-binding fragment thereof, of the present invention specifically binds to a specific domain of the catalytic subunit alpha of protein kinase A, thereby significantly increasing its affinity compared to the conventional catalytic subunit alpha antigen of protein kinase A. It was confirmed that cancer patients can be diagnosed. Accordingly, by using the antibody or antibody fragment of the present invention, the catalytic subunit alpha of protein kinase A or an autoantibody therefor can be specifically detected at the protein level in the future to enable accurate diagnosis of cancer. has industrial applicability.

Claims (20)

1. An antibody or antibody fragment that specifically binds to the catalytic subunit alpha of protein kinase A that recognizes a polypeptide selected from the group consisting of the amino acid sequence shown in SEQ ID NOs: 1 to 5 as epitope.
2. According to claim 1,

   The antibody or fragment of the antibody comprises a heavy chain complementarity determining region (CDR)1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7, a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9, and a heavy chain variable region comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11; and a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, a light chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 17 An antibody or fragment of an antibody, characterized in that it comprises a light chain variable region comprising a light chain CDR3 comprising a.
3. According to claim 1,

   The antibody or antibody fragment comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 19; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 21, an antibody or antibody fragment.
4. According to claim 1,

   Wherein the antibody is selected from the group consisting of IgG, IgA, IgM, IgE and IgD, antibody or antibody fragment.
5. According to claim 1,

   The antibody fragment is selected from the group consisting of diabody, Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv, dsFv, Fd, Fd' and scFv, characterized in that the antibody or antibody snippet.
6. A polynucleotide encoding the antibody or fragment of any one of claims 1 to 5.
7. 7. The method of claim 6,

   The polynucleotide is a polynucleotide, characterized in that it comprises one or more sequences selected from the group consisting of nucleotide sequences selected from the group consisting of SEQ ID NOs: 22 to 33.
8. 7. The method of claim 6,

   The polynucleotide is a polynucleotide, characterized in that it comprises one or more sequences selected from the group consisting of nucleotide sequences selected from the group consisting of SEQ ID NOs: 34 to 37.
9. A recombinant expression vector comprising the polynucleotide of claim 6.
10. A cell transformed with the recombinant expression vector of claim 9 .
11. (a) transforming the host cell with the recombinant expression vector of claim 9;

    (b) culturing the transformed host cell to produce the antibody or antibody fragment; and

    (c) obtaining the antibody or fragment of the antibody produced in the host cell;

    A method for producing an antibody or fragment of an antibody that specifically binds to the catalytic subunit alpha of protein kinase A.
12. A composition for diagnosing cancer comprising the antibody or antibody fragment of any one of claims 1 to 5.
13. 13. The method of claim 12,

    The cancer is colon cancer, colorectal cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, villous cancer, ovarian cancer, breast cancer, thyroid cancer, brain cancer, both A composition for diagnosing cancer, characterized in that it is selected from the group consisting of cervical cancer, malignant melanoma, lymphoma, aplastic anemia and hematologic cancer.
14. A kit for diagnosing cancer comprising the composition of claim 12.
15. Claims 1 to 5, comprising the step of detecting the catalytic subunit alpha of protein kinase A in a biological sample isolated from an individual suspected of cancer using the antibody or antibody fragment of any one of claims 1 to 5 through an antigen-antibody reaction A method of providing information for the diagnosis of cancer.
16. An autoantibody to the catalytic subunit alpha of protein kinase A is detected through an antigen-antibody reaction in a biological sample isolated from an individual suspected of cancer using the antibody or antibody fragment of any one of claims 1 to 5 A method for providing information for diagnosing cancer, comprising the step of:
17. Use of a composition comprising the antibody or antibody fragment of any one of claims 1 to 5 for diagnosing cancer.
18. Use of the antibody or fragment of any one of claims 1 to 5 for the production of a diagnostic agent for cancer.
19. The catalytic subunit alpha of protein kinase A or an autoantibody thereto from a biological sample isolated from an individual suspected of cancer using the antibody or antibody fragment of any one of claims 1 to 5 through an antigen-antibody reaction A method for diagnosing cancer, comprising the step of detecting.
20. The catalytic subunit alpha of protein kinase A or an autoantibody thereto from a biological sample isolated from an individual suspected of cancer using the antibody or antibody fragment of any one of claims 1 to 5 through an antigen-antibody reaction A method of predicting the risk of developing cancer, comprising the step of detecting.

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